Non-optimal codon usage in the gene for circadian clock genes leads to a fitness advantage for cyanobacteria grown at cool temperatures.

In the 1960s, Marshall Nirenberg and H. Gobind Khorana broke the genetic code by demonstrating that groups of three bases in DNA (codons) specify the incorporation of a specific amino acid into a protein and identifying which amino acid was associated with each codon. Since there are 64 possible codons and only 20 amino acids, nearly all amino acids are designated by more than one codon. This degeneracy in the DNA code helps to mitigate the effects of mutations; however other possible functions have remained obscure. Every species has its own pattern of preferred codon usage, meaning that multiple codons for the same amino acid are used at different frequencies, with usually one codon predominating over the others. The mechanisms behind this codon selectivity are not fully understood; however, genes that are highly expressed tend to display preferential usage of codons corresponding to the most abundant tRNA for a given amino acid. This pattern of “optimal” codon usage facilitates efficient transcription and reduces the chance of error. Thus, VICB member Carl Johnson and his laboratory were surprised to find that, in the cyanobacterium Synechococcus elongatus, the genes encoding the highly expressed circadian clock proteins displayed non-optimal codon usage, resulting in a fitness advantage for the bacteria under cool temperature conditions [Y. Xu, et al. (2013) Nature, published online February 17, DOI:10.1038/nature11942].

Although cyanobacteria are very simple organisms, they are capable of carrying out oxygenic photosynthesis as well as oxygen-sensitive nitrogen fixation. These are two incompatible processes, necessitating that the bacteria devise a mechanism to physically or temporally separate them. The solution was to establish a circadian rhythm, allowing photosynthesis to occur during the day when light is available and nitrogen fixation to occur at night. Cyanobacteria are the most primitive organism to exhibit a biochemical circadian oscillation.

The circadian clock of S. elongatus is regulated by three proteins, KaiA, KaiB, and KaiC, which are encoded by a single operon containing a monocistronic gene, kaiA, and a discistronic gene, kaiBC. The KaiC protein, which exists as a hexameric complex in solution, has both an autokinase and an ATPase activity. Incubation of the three Kai proteins in vitro with ATP establishes a rhythmic cycle of phosphorylation-dephosphorylation of KaiC that is modulated by KaiA and KaiB (Figure 1). This cycling of the phosphorylation state of KaiC, which occurs with a period of 24 h even in the absence of a light-dark cycle, forms the foundation for the S. elongatus circadian clock.

Figure 1. The KaiC protein (blue) comprises a C1 and a C2 domain with C-terminal “tentacles”. It exists as a hexamer in solution. At the beginning of the cycling reaction, KaiA (pink) repeatedly and rapidly interacts with KaiC’s C-terminal tentacles, initiating and propagating the phosphorylation phase. When KaiC becomes hyperphosphorylated (red spheres), it first binds KaiB (green) stably. Then, the KaiB•KaiC complex binds KaiA, sequestering it from further interaction with KaiC’s tentacles. At that point, KaiC initiates dephosphorylation. When KaiC is hypophosphorylated, it releases KaiB and KaiA, thereby launching a new cycle. Image kindly provided by Carl Johnson. Copyright, Carl Johnson.

Because the kai genes are among the most abundantly expressed in S. elongatus, the Johnson lab initially hypothesized that they would exhibit optimal codon usage. They found, however, that the kaiBC transcript actually displays below average optimal codon usage based on the codon adaptation index. To better understand the impact of this unexpected finding on cyanobacterial metabolism, the investigators constructed two mutant strains. In the optKaiB strain, they replaced the suboptimal codons in the kaiB gene with codons more closely resembling those found in highly expressed genes. In the optkaiBC strain, the “optimization” was extended to include the 5′ region of the kaiC gene as well. The investigators had hypothesized that nonoptimal codon usage in the kaiBC gene would be required for normal rhythmicity. However, when they grew the wild-type, optKaiB, and optKaiBC strains at the optimal growth temperature of 30oC, they found no effect of optimization on circadian clock function. In fact, the optimized strains maintained rhythmicity over a wide range of temperatures, while rhythmicity was lost in the wild-type cyanobacteria at cooler temperatures in the range of 18 to 20oC (Figure 2).

Figure 2. The rhythmic cycles of the S. elongatus circadian clock could be monitored by expressing a fluorescent protein under control of the kaiBC promoter. The luminescence of cells grown in constant light indicated the ability of the clock to maintain its rhythm in the absence of an external light-dark cycle. Wild-type cells and both optimized strains maintained rhythmicity similarly at warm temperatures. However, culture of wild-type cells at cool temperatures led to a loss of rhythmicity, which was maintained longer in the optimized strains. Reproduced by permission from Macmillin Publishers Ltd. from Y. Xu, et al. (2013) Nature, published online February 17, DOI:10.1038/nature11942. Copyright 2013.

Codon optimization had resulted in increased expression of the KaiB and KaiC proteins, suggesting a possible explanation for the retention of rhythmicity at cool temperatures in the optKaiB and optKaiBC strains. Consistently, inducible overexpression of KaiB and KaiC together had the same effect as gene optimization, though rhythmicity was totally lost at very high levels of protein expression, or when KaiB was overexpressed without KaiC.

Prior work has indicated that at optimal temperatures when growth rate is high, the circadian clock offers a clear survival advantage to cyanobacteria. One might therefore assume that the superior ability to maintain rhythmicity at cool temperatures would offer a similar advantage to the optimized strains. The Johnson group tested this hypothesis by measuring growth rates for wild-type S. elongatus, the optKaiBC strain, and two strains (CLAb and CLAc) bearing mutations in KaiC that result in markedly dampened circadian rhythms. They found that at 30oC, the optimized strains exhibited a slight growth advantage, while the KaiC mutant strains grew more slowly than the wild-type cells. However, as the temperature was lowered, optKaiBC lost its advantage and, in fact, became the most slowly growing strain. In contrast, as the temperature decreased, the growth rates of CLAb and CLAc approached and even exceeded that of the wild-type strain (Figure 3). These unexpected results suggest that loss of circadian rhythmicity imparts a growth advantage to S. elongatus at cool temperatures, which are associated with slow growth rates. It should be noted that the temperatures used for these experiments are well within the range of those that S. elongatus experiences in its normal environment. Thus, the investigators conclude that the use of non-optimal codons in the kaiBC gene is, in fact, a mechanism that allows the cyanobacterium to turn off its circadian clock when doing so offers a survival advantage. Clearly, this discovery opens a new avenue of thought regarding the role of optimum codon usage in the post-transcriptional regulation of gene expression.

Figure 3. Growth of wild-type S. elongatus as compared to the optKaiBC strain, and two strains bearing mutations in KaiC (CLAb and CLAc). The KaiC mutant strains have markedly dampened circadian clocks at all temperatures, while optKaiBC shows rhythmicity at all temperatures, and the wild-type strain exhibits rhythmicity at high temperatures, but not at lower ones. Reproduced by permission from Macmillin Publishers Ltd. from Y. Xu, et al. (2013) Nature, published online February 17, DOI:10.1038/nature11942. Copyright 2013